Immunoglobulin G genetic variation can confound assessment of antibody levels via altered binding to detection reagents

Abstract Objectives Amino acid variations across more than 30 immunoglobulin (Ig) allotypes may introduce structural changes that influence recognition by anti‐Ig detection reagents, consequently confounding interpretation of antibody responses, particularly in genetically diverse cohorts. Here, we assessed a panel of commercial monoclonal anti‐IgG1 clones for capacity to universally recognise two dominant IgG1 haplotypes (G1m‐1,3 and G1m1,17). Methods Four commercial monoclonal anti‐human IgG1 clones were assessed via ELISAs and multiplex bead‐based assays for their ability to bind G1m‐1,3 and G1m1,17 IgG1 variants. Detection antibodies were validated against monoclonal IgG1 allotype standards and tested for capacity to recognise antigen‐specific plasma IgG1 from G1m‐1,3 and G1m1,17 homozygous and heterozygous SARS‐CoV‐2 BNT162b2 vaccinated (n = 28) and COVID‐19 convalescent (n = 44) individuals. An Fc‐specific pan‐IgG detection antibody corroborated differences between hinge‐ and Fc‐specific anti‐IgG1 responses. Results Hinge‐specific anti‐IgG1 clone 4E3 preferentially bound G1m1,17 compared to G1m‐1,3 IgG1. Consequently, SARS‐CoV‐2 Spike‐specific IgG1 levels detected in G1m1,17/G1m1,17 BNT162b2 vaccinees appeared 9‐ to 17‐fold higher than in G1m‐1,3/G1m‐1,3 vaccinees. Fc‐specific IgG1 and pan‐IgG detection antibodies equivalently bound G1m‐1,3 and G1m1,17 IgG1 variants, and detected comparable Spike‐specific IgG1 levels between haplotypes. IgG1 responses against other human coronaviruses and influenza were similarly poorly detected by 4E3 anti‐IgG1 in G1m‐1,3/G1m‐1,3 subjects. Conclusion Anti‐IgG1 clone 4E3 confounds assessment of antibody responses in clinical cohorts owing to bias towards detection of G1m1,17 IgG1 variants. Validation of anti‐Ig clones should include evaluation of binding to relevant antibody variants, particularly as the role of immunogenetics upon humoral immunity is increasingly explored in diverse populations.


INTRODUCTION
Reliable antibody-based detection reagents are fundamental to biomedical research, and antibody detectors targeted against immunoglobulin (Ig) molecules are of particular importance in immunological and clinical research.However, insufficient validation or inappropriate use of commercial antibody-based tools continues to contribute substantially to the reproducibility crisis pervasive within biomedical research. 1,2As such, it is critical that investigators are aware of the importance of thoroughly validating anti-IgG reagents for use in desired applications.
Anti-Ig antibodies are raised against the constant heavy chain (C H ) of an antibody molecule (Figure 1a) which, in theory, should render these reagents specific to distinct antibody isotypes (IgA, IgD, IgE, IgG and IgM) or subclasses (IgA1-2 and IgG1-4).However, despite its name, the antibody constant region contains a diverse array of single nucleotide polymorphisms (SNPs) that introduce considerable variation at the amino acid level.This sequence diversity has been most comprehensively characterised for IgG and IgA, with IgG3, followed by IgG1, demonstrating the most extensive variation. 3hese Ig polymorphisms were initially termed 'allotypes' following identification in 1956 via rabbit anti-sera produced by animals inoculated with serum from an immunogenetically distinct counterpart. 4Almost three decades later, monoclonal anti-allotype antibody detectors were developed. 5Given the immunogenic nature of allotypes, it is not surprising that these alleles underlie variable interactions between immunoglobulins and their respective anti-isotype detection reagents.Although individual allotypic markers are typically defined by only one or two amino acid substitutions, these substitutions may alter protein-protein interactions by mutating the epitope recognised by the detection antibody, interfering with binding via steric hindrance, or by inducing conformational changes at the binding site of the detection antibody, even if this epitope is distant from the position of the substituted amino acids. 6Nevertheless, despite the initial identification of allotypes via serological methods, thorough validation of binding by antibody detection reagents to different Ig allotypes is not routinely performed.
IgG1 is the most abundant immunoglobulin in humans, constituting approximately 65% of IgG. 7,8gG1 is important for protection against infectious agents 3,7,9,10 and cancer, 11,12 while also implicated in autoimmune diseases. 13,14Four IgG1 allotypic markers (G1m) have been defined: G1m1, G1m2, G1m3 and G1m17 (Figure 1b).G1m3 and G1m17 are antithetical markers, while the alternate alleles for G1m1 and G1m2 allotypes are denoted as the absence of the marker (nG1m1/G1m-1 and nG1m2, respectively).As immunoglobulin allotypes are inherited in a Mendelian fashion, IgG1 haplotypes tend to cluster ethnically and geographically.For example, the G1m-1,3 haplotype is dominant in individuals of European descent, while the G1m1,17 haplotype is more prevalent, reaching frequencies over 80%, within African and Asian populations as well as American and Australian First Nations peoples. 3,15,16ere, we demonstrate that certain commercial anti-IgG1 antibodies display variable binding to G1m-1,3 and G1m1,17 IgG1 variants.We show that a detection antibody targeting the IgG1 hinge region preferentially binds G1m1,17 over G1m-1,3 variants, while IgG1 and pan-IgG clones raised against the Fc portion bind G1m-1,3 and G1m1,17 IgG1 equivalently.These data emphasise the importance of thoroughly validating the suitability of antibody detection reagents for the characterisation of humoral responses, particularly in small clinical cohorts comprising genetically diverse individuals.

Anti-IgG1 clone influences antigen-specific IgG1 levels detected in patient samples
Following the observation that the 4E3 anti-IgG1 clone, but not the MTG1218, HP6001 and HP6069 clones, differentially bind G1m-1,3 and G1m1,17 allotype mAb standards (Figure 1c-f), we next aimed to determine whether the detector clone influenced quantification of antigen-specific IgG1 levels in a small clinical cohort.Using a custom Luminex bead-based multiplex assay, IgG1 levels against SARS-CoV-2 and influenza were assessed in homozygous G1m-1,3/G1m-1,3 and G1m1,17/G1m1,17 BNT162b2 vaccinees using each of the four anti-IgG1 detection reagents and compared between haplotypes.
Correlation of IgG1 levels in BNT162b2 vaccinated individuals measured using each of the 4E3, HP6001, HP6069 or MTG1218 anti-IgG1 clones, indicated similar binding behaviour of HP6001, HP6069 and MTG1218 (r = 0.96-0.98),while 4E3 showed limited correlation with any other anti-IgG1 clone (r = 0.32-0.38; Figure 2f, g).Indeed, correlation of IgG1 responses measured with 4E3 and each of HP6001, HP6069 or MTG1218 resulted in two distinct clusters (Figure 2f, g).One cluster corresponded to G1m1,17/G1m1,17 individuals for whom reasonable correlation between 4E3 and other anti-IgG1 clones was observed.The alternate cluster corresponded to G1m-1,3/G1m-1,3 individuals for whom no correlation between 4E3 and other clones was evident.These correlations reinforce the conclusion that 4E3 does readily not bind the G1m-1,3 IgG1 variant, as suggested by the EC 50 s calculated for this detector (Figure 1c), as well as comparisons between IgG1 responses measured using 4E3 and alternative anti-IgG1 clones (Figure 2a-e).

Anti-IgG1 clone 4E3 may confound assessment of vaccination and infection induced antibodies from clinical cohorts
The potential for hinge-specific anti-IgG1 detector clone 4E3 to artificially bias antibody levels measured for clinical cohorts was next demonstrated in BNT162b2 vaccinated and COVID-19 convalescent cohorts that included G1m-1,3/G1m1,17 heterozygous individuals.Given that responses measured with the Fc-specific clones HP6001 and HP6069, as well as MTG1218, strongly correlated, only 4E3 and HP6001 were used for further comparison.

DISCUSSION
Antibody detection reagents frequently contribute to irreproducible studies within biomedical research. 1,2This is typically a ramification of antibody cross-reactivity or inappropriate use of antibody reagents in applications for which they have not been validated.Alternatively, as demonstrated here in the case of IgG allotypes, altered binding may occur when SNPs interfere with the capacity of detector reagents to recognise variants of the canonical protein isoform.
We demonstrate that the G1m1,17 IgG1 variant is preferentially bound by the hinge-specific anti-IgG1 detection antibody clone 4E3.Notably, the G1m3/G1m17 marker likely drives the IgG1 haplotype-associated differential binding by 4E3.In contrast to Fc-specific HP6001 and HP6069 anti-IgG1 clones (the epitope of MTG1218 remains proprietary) as well as Fc-specific pan-IgG clone JDC-10, 4E3 binds the hinge region.The G1m3/17 marker is located distally along the C H 1 region at position 214, close to the IgG1 hinge (Figure 1b).The lysine (G1m17) to arginine (G1m3) substitution introduces a guanidine head group in place of the amino head group.Although lysine and arginine are both basic amino acids with similar chemical properties, the additional side chain present in arginine may impair binding of G1m-1,3 IgG1 by hinge-specific 4E3.However, the possibility of a singular or combinatorial influence of the G1m1/nG1m1 allotype upon detection of IgG1 by 4E3 cannot be excluded.Furthermore, the influence of the G1m2/nG1m2 allotype upon anti-IgG binding remains to be determined.
Assessment of Ig detection reagent binding for universal allotype compatibility is essential in immunogenetics-focused investigations.][20][21][22][23][24] However, a compelling mechanism for this phenomenon has yet to be elucidated.We demonstrate that when assay readouts directly depend upon binding by anti-Ig detection reagents (e.g.ELISA, radioimmunoassay, radial immunodiffusion and variations thereof), equivalent detection antibody binding to all represented haplotypes should be confirmed prior to assessing immunogenetic influences upon antibody responses.
Promisingly, studies of allotype-associated immune response variations have not been restricted to quantitative serology-based assays susceptible to the confounding influence of inadequately validated detection antibodies.Enhanced disease protection 23,25 and functional immune responses 26 have also been associated with various IgG haplotypes, implying that associations observed between IgG allotypes and altered antibody levels cannot be discounted, but should be confirmed with thoroughly validated detection reagents.As such, this topic warrants meticulous further investigation.
Awareness of the possible confounding influence of allotypes upon anti-Ig detection antibody binding is of particular importance when assessing humoral responses in rare and unique clinical cohorts which typically rely on small sample sizes, especially when genetically diverse participants are recruited.In the likely instance that Ig allotypes are not equally represented across all study groups, inadequate anti-Ig detection reagent validation may result in antibody responses for impacted experimental groups being artificially inflated if the selected detection antibody discriminates between allotypes.Critically, as Ig variants are increasingly described in populations historically underrepresented in the biomedical literature 27   immunogenetics upon personalised vaccine responses heightens, 3 the potential for host genetic variation to introduce experimental artefacts should be afforded greater consideration.Accordingly, investigators should thoroughly assess for the absence of any possible confounding interactions between novel alleles and antibody detection reagents.Such validation is essential for further work suggested by the present study as the influence of IgG1 allotypes upon SARS-CoV-2 antibody responses remains to be fully characterised in cohorts with more extensive coverage of homozygous and heterozygous G1m allelic combinations.
Here we have demonstrated the influence of G1m-1,3 and G1m1,17 haplotypes upon binding of monoclonal anti-IgG1 detection reagents to human IgG1.][30] Furthermore, investigators should be aware that the phenomenon of allotype-associated detection antibody incompatibility likely exists between variants of other Ig isotypes and subclasses and their respective detection reagents.Such 'serological blind spots' have been observed for IgG3 allotypes, 31 consistent with the highly polymorphic nature of this subclass, 3 but have also been observed within the more conserved IgG4 subclass. 31Nevertheless, other investigators assessing this phenomenon have not detected inconsistencies in binding to different allotypes within alternative panels of detection reagents, observing instead only cross-reactivity between IgG subclasses. 32n conclusion, we highlight the critical importance of thoroughly validating commercial antibody detection reagents used for serological characterisation of clinical cohorts, particularly those that include immunogenetically diverse participants.

METHODS Monoclonal IgG1 allotype standards and anti-IgG antibody detection reagents
Four monoclonal IgG1 allotype antibodies (allotype mAb standards) were used as target antigens to quantify the half maximal effective concentration (EC 50 ) of a panel of commercial monoclonal mouse anti-human IgG1 detection reagents.Both kappa and lambda light chain variants of the G1m-1,3 and G1m1,17 allotype mAb standards were used to confirm that the sequence variations between kappa and lambda light chains did not influence binding of anti-IgG1 detectors.All four allotype mAb standards were recombinant antibodies with specificity for green fluorescent protein.The details of the four allotype mAb standards are tabulated in Supplementary table 1.
Four monoclonal anti-human IgG1 antibody detection reagents (clones 4E3, HP6001, HP6069 and MTG1218) were tested against the four allotype mAb standards and used to assess antibody levels in patient plasma.One monoclonal anti-human pan-IgG antibody detection reagent (clone JDC-10) was used to assess antibody levels in patient plasma.The details of the five anti-IgG antibody detection reagents are tabulated in Supplementary table 2.

DNA extraction, polymerase chain reaction (PCR) and sequencing
G1m1/nG1m1 and G1m3/G1m17 typing of study participants was performed via PCR, as previously described, 35,36 with minor modifications.Genomic DNA was extracted from granulocytes using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany; 51104) according to the manufacturer's instructions.Amplification of the human C H 1 and C H 3 domains of IGHG1 was performed using the AccuPrime Taq DNA Polymerase, High Fidelity system (Thermo Fisher Scientific, 12346094) prepared with 150 ng DNA template, 3 lL 109 AccuPrime PCR buffer II, 5 units per lL enzyme and 0.25 lM each primer, brought to a total volume of 30 lL in nuclease-free water.Initial denaturation was performed at 94 °C for 30 s followed by 35 cycles of denaturation at 94 °C for 25 s, annealing at 62 °C for 25 s and extension at 72 °C for 50 s, with a final extension at 72 °C for 7 min.Dual direction sequencing of PCR products was performed by the Australian Genome Research Facility (AGRF, Melbourne, VIC, Australia).Geneious Prime version 2023.2.1 was used for sequence analysis and genotypes were manually called.

Statistical analysis
Prism GraphPad version 10.1.0(GraphPad Software, San Diego, CA, USA) was used to develop graphs and perform the statistical analyses described in the figure captions.Half maximal effective concentration (EC 50 ) of each anti-IgG1 detector was determined using a four-parameter nonlinear regression model describing the relationship between agonist concentration and response.

Figure 1 .
Figure 1.(a) Structure of an IgG1 antibody.Immunoglobulins comprise two fragment antigen-binding (Fab) regions and a fragment crystallisable (Fc) region connected by a flexible hinge.Two constant heavy (C H ) and two constant light (C L ) chains are connected via disulphide bridges, which, along with two variable heavy (V H ) and two variable light (V L ) chains, form an ~150 kDa molecule.The C H chain comprises three structural regions (C H 1-C H 3) as well as the flexible hinge.(b) IgG polymorphisms giving rise to allotypes are located within the C H 1 and C H 3 regions of IgG1.EC 50 s of (c) 4E3, (d) HP6001, (e) HP6069 and (f) MTG1218 anti-IgG1 clones for kappa and lambda variants of G1m-1,3 and G1m1,17 allotype mAbs.Measurements were performed in triplicate and mean values AE SEM are indicated.Curves were fitted using a fourparameter nonlinear regression.Mann-Whitney U-tests were performed between responses against G1m1,17 (kappa and lambda) and G1m-1,3 (kappa and lambda) allotype mAb standards within each concentration of 4E3 anti-IgG1 detection reagent.P < 0.01 (**); P < 0.05 (*).
and as interest in the role of ª 2024 The Authors.Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc. 2024 | Vol. 13 | e1494